In order to delineate the mechanism of signal transduction for growth factor receptors at the initial stage of binding, we focused on defining the tertiary structure of the extracellular domain of epidermal growth factor (EGF) receptor. Using conditioned medium from a bioreactor containing A431 cells grown on hollow fibers, we purified to homogeneity of a glycoprotein having a mass of ca. 115 kilodaltons and specifity towards a monoclonal antibody raised against the EGF receptor. Using a two step purification scheme (Q-Sepharose and Sepharyl S-300 chromatography) better than 90% pure soluble EGF receptor was obtained. The IEF gel analysis of this glycoprotein isolated from after 2 to 15 months of continuous culturing A431 cells in the bioreactor indicated the presence of significant heterogeneneity with respect to glycosylation. The analysis indicated the presence of different glycosylated forms with pI of 7.0 to 5.5. CD spectroscopic analysis of the secreted EGF receptor indicate the presence of a-helix, b-sheet, and coil structures. CD spectra further support unfolding of the molecule upon addition of 4M guanidine-HCI; the extent of this denaturation indicates greater stability of b-sheet over a- helical structure. Lastly, EGF binding to this soluble receptor induced a CD spectral change which supports the notion that a ligand binding to a receptor would induce tertiary structure change. In order to determine the tertiary structure of the receptor and its complex with EGF, the crystallization of the soluble receptor and its complex with EGF and TGF-a is currently being attempted.